Abstract

Background

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective loss of motor neurons (MN) in the brain stem and spinal cord. Intracellular disruptions of cytosolic and mitochondrial calcium have been associated with selective MN degeneration, but the underlying mechanisms are not well understood. The present evidence supports a hypothesis that mitochondria are a target of mutant SOD1-mediated toxicity in familial amyotrophic lateral sclerosis (fALS) and intracellular alterations of cytosolic and mitochondrial calcium might aggravate the course of this neurodegenerative disease. In this study, we used a fluorescence charged cool device (CCD) imaging system to separate and simultaneously monitor cytosolic and mitochondrial calcium concentrations in individual cells in an established cellular model of ALS.

Conclusion

In this study, by fluorescence measurement of cytosolic calcium and using simultaneous [Ca2+]i and [Ca2+]mito measurements, we are able to separate and simultaneously monitor cytosolic and mitochondrial calcium concentrations in individual cells an established cellular model of ALS. The primary goals of this paper are (1) method development, and (2) screening for deficits in mutant cells on the single cell level. On the technological level, our method promises to serve as a valuable tool to identify mitochondrial and Ca2+-related defects during G93A-mediated MN degeneration. In addition, our experiments support a model where a specialized interplay between cytosolic calcium profiles and mitochondrial mechanisms contribute to the selective degeneration of neurons in ALS.

Background

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by a selective loss of motor neurons (MNs) in the brain stem, the spinal cord and the motor cortex, leading to progressive weakness, muscle atrophy with eventual paralysis, and death. Approximately 5–10% of ALS cases are familial [1, 2]. A decade ago, researchers discovered missense mutations in the gene encoding the Cu/Zn superoxide dismutase 1 (SOD1) in subsets of familial cases; approximately 20% of familial ALS (fALS) is caused by mutations in SOD1 with high inter-subject variation of progression, including the point mutation G93A [3–7]

Multiple cascades have been implicated in the motor neuron death pathway, including mitochondrial dysfunction and deformities [8–15]; complex I, III, and IV abnormalities [16–19]; mitochondrial alteration, increase in Ca2+ uptake and increase of cytosolic Ca2+ concentration [6, 20], oxidative stress [21]; and glutamate excitotoxicity [22–29]. Alteration and disruption of calcium homeostasis and metabolism [30–40] has also been reported.

The present evidence supports a hypothesis that mitochondrial dysfunction acts with oxidative stress to cause abnormal neurodegeneration via calcium-mediated MN injury. Oxidative stress may lead to increased intracellular calcium, which leads to increased nitric oxide and peroxynitrite formation [21]. Glutamate excitotoxicity may disrupt intracellular calcium homeostasis and reactive oxygen species (ROS) production [27], which may be promoted by oxidative stress as glutamate transporters are particularly susceptible to disruption by oxidants, and oxidative modifications to the transporter have been reported in ALS and the mtSOD1G93A mouse model [24, 21]. The etiology is likely to be multifactorial because ALS involves the interplay of several mechanisms to initiate disease and propagate the spread of motor neuron cell death [36, 38, 39].

Mitochondrial Ca2+ uptake responds dynamically and sensitively to changes in cytosolic Ca2+ levels and plays a crucial role in sequestering the large Ca2+ loads induced by FCCP-evoked Ca2+ influx [41]. The excessive influx of Ca2+ into mitochondria may result in mitochondrial dysfunction. Prominent and sustained mitochondrial depolarization follows intense ion channel receptor stimulation and closely parallels the incidence of neuronal death [42]. Substantial Ca2+ can be accumulated in mitochondria as a result of overloading the matrix with Ca2+; this disrupts the structural and functional integrity of the organelle. Hence, mitochondria may be a critical intracellular target of injury after intense Ca2+ channel stimulation and, in this way, may act as a link between massive Ca2+ influx and mitochondria-mediated neurotoxicity by mtSOD1G93A. However, the precise relationship between Ca2+ influx, cytosolic Ca2+ increase, and mitochondrial Ca2+ uptake remains obscure.

Culture systems, such as slice culture or primary cultures of MN, have proven to be valuable tools in the physiological and biochemical characterization of ALS-related pathology [43–45]. Still unknown is whether the presence of mtSOD1G93A causes morphological mitochondrial abnormalities when expressed at physiological levels, whether the source specificity of mitochondrial Ca2+ sequestration and spatiotemporal properties of cytosolic Ca2+ ([Ca2+]i) signaling in cells transfected with wild-type and G93A-mutant SOD1 varies at physiological levels, and if there are functional consequences of changes in mitochondrial function on Ca2+ homeostasis in the presence of mtSOD1G93A. To elucidate the underlying molecular events and cellular alterations involved in oxidative stress induced by the aberrant Cu-Zn chemistry and the roles of impaired Ca2+ handling and oxidative stress induced by ROS in fALS, we used SH-SY5Y neuroblastoma cells transfected with the G93A mutant form of SOD1 typical for fALS (SH-SY5YG93A or G93A) and SH-SY5Y cells transfected with wild-type (WT) human SOD1 (SH-SY5YWT or WT) and mimic the situation present in heterozygous patients, previously established as an in-vitro cell culture model of ALS, resembling the situation of heterozygous patients [6, 46]. This particular mutation was chosen because it does not affect the activity of SOD1; previously it was shown that SH-SY5Y cells expressing G93A exhibit increased intracellular ROS [47]. The effect of the continuous expression of WT or G93A on mitochondrial morphology and Ca2+ signaling were compared. In addition, the consequences and impact of protonophore FCCP and other organelle-specific drugs on mitochondrial vulnerability and Ca2+ homeostasis were studied.

Methods

SH-SY5YWT and SH-SY5YG93A transfected neuroblastoma cell cultures

An in-vitro model to study the cellular alterations associated with mutations of SOD1 was constructed by transfection of the human neuroblastoma cell line SH-SY5Y with G93A-SOD1 [6]. The cell types that we have used in our study are well characterized and have been used as cellular model system for motoneuron disease by several groups [6, 48–50]. The transfected cell lines have relatively low levels of mtSOD/wtSOD and are therefore an attractive model system for the human disease [50]. Upon expression of fALS – SOD1, these cells show several features typical of neurons of both ALS patients and transgenic mice, such as mitochondrial alteration, increased ROS levels and increased cytosolic calcium concentration [see references in [49]].

Changes in [Ca2+]i were monitored by the excitation of Fura-2 AM at 360 nm and 390 nm; emitted light was directed to a dichroic mirror by a beam splitter at 425 nm. In the case of non-transfected SH-SY5Y cells, the excitation of Fura-2 AM was performed at 356 nm and 385 nm and the emitted light was directed to a dichroic mirror with mid-reflection at 425 nm. In some experiments, the light was filtered by a band pass filter of 505–530 nm [See Additional file 1]. Since the background fluorescence is not clearly determinable in AM loaded cells, we regarded the calculated Ca2+ concentrations as an approximation of the real concentration [19]. Therefore, for ester-loaded cells, changes in [Ca2+]i are given as changes in the Fura-2 ratio (360/390 nm).

Fura-2 (Kd ~ 0.2 μM) was monitored by a computer-controlled monochromator (Polychrome II, TILL Photonics, Germany), which was connected to an Axioscope microscope (Zeiss, Goettingen, Germany) via quartz fiber optics and a minimum number of optical components for maximum fluorescence excitation (objective Achroplan W 63x, 0.9W). The apparatus was equipped with a CCD camera system [51, 52], which displayed 12-bit dynamics (PCO, Germany), and an A/D converter with a 12.5 MHz sampling rate; the binning was set to 4 × 4 and the exposure time was 30–80 ms. Calcium changes in defined regions of interest (ROIs) were monitored online using TILL Vision Software V3.3 (TILL Photonics, Martinsried, Germany). The measured fluorescence ratio at the selected wavelengths was used to calculate the [Ca2+]i and was represented as F/F0. Further analysis was performed off-line with the IGOR software (Wavemetrics, Lake Oswego, OR, USA) and Origin software, version 7.5.

Simultaneous recording of changes in [Ca2+]i and [Ca2+]mito

Simultaneous fluorometric measurements of [Ca2+]i and mitochondrial calcium ([Ca2+]mito) were performed utilizing calcium-sensitive fluorescent dyes (Fura-2 AM for cytosolic calcium and Rhod-2 AM for mitochondrial calcium). SH-SY5Y cells expressing either WT or G93A were attached to glass cover slips and loaded initially with 5 μM Fura-2 AM at 37°C for 30 min as described above. Subsequently, cells were washed with RPMI-1640 for 20 min and further incubated with 10 μM Rhod-2 AM for 20 min at 37°C (5% CO2 and 95% O2). The coverslips were rinsed with RPMI-1640 and further incubated for 20 min at 37°C to allow for complete de-esterification.

To clearly separate and simultaneously monitor the dynamic [Ca2+]i and [Ca2+]mito profiles with a temporal resolution in the millisecond time domain, we utilized a computer-controlled monochromator that rapidly switched between excitation wavelengths of 390 nm (Fura-2) and 550 nm (Rhod-2) and collected wavelengths above 565 nm for both excitation conditions. Schematic representation of the CCD-imaging setup used for conventional calcium imaging and simultaneous imaging of [Ca2+]i and [Ca2+]mito signals in the cell line preparation are illustrated in Additional file 1 [See Additional file 1]. For kinetic data analysis, only Fura-2 and Rhod-2 signals from pre-defined regions of cells were used.

The accumulations of mitochondrial and other cell organelles calcium were inhibited by cell organelles specific drug modulators. First, drugs were used, including KCl (K+), depolarisation stimulus; FCCP, a protonophore; and oligomycin (oligo), a F1, F0-ATPase blocker preventing maintenance of the proton gradient, which disrupt the mitochondrial membrane potential (ΔΨm) [53–56]. Secondly, by thapsigargin (thapsi) an extremely tight binding inhibitor of intracellular Ca2+ pumps, which raises cytosolic calcium concentration by blocking the ability of the cell to pump calcium into the sarcoplasmic and endoplasmic reticulum which causes these stores to become depleted [57]. Store-depletion can secondarily activate plasma membrane calcium channels, allowing an influx of calcium into the cytosol. Thirdly, by caffeine, a specific activator of Ca2+ influx from ryanodine receptor (RyR)-dependent Ca2+ stores [58].

Statistical analysis

Each cover slip was used for a single experiment and included more than 4 cells for each imaging experiment. Unless otherwise indicated, values in the text are given as mean ± standard deviation (SD) and the error bars indicate SD too. All values represent at least three separate experiments. Significance was calculated using the two tailed unpaired Student t-test. A p-value < 0.05 was consider statistically significant. Five point smoothing was performed in the case of simultaneous [Ca2+]i and [Ca2+]mito signals to remove the noise.

Chemical induction of calcium release

Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), thapsigargin, and oligomycin were purchased from Sigma-Aldrich Chemie (Deisenhoff, Germany). FCCP was bath applied at a dose of 2 μM. A comparative analysis of the Ca2+ influx in WT and G93A transfected cells was done in the presence of FCCP. For these experiments the SH-SY5Y cells were loaded with 5 μM Fura-2 AM through the incubation method and [Ca2+]i transients were triggered once Fura-2 AM concentration was equilibrated after washing. Ca2+ release from intracellular stores by inhibition of the sarcoplasmic/endoplasmic reticulm Ca2+-dependent ATPase pump and inhibition of F1, F0-ATP Synthase was done by intervention of thapsi, and oligo, respectively.

For the investigation of endoplasmic reticulum (ER)-dependent Ca2+ release, caffeine has been established as a specific activator of Ca2+ influx from the RyR-dependent Ca2+ stores [58]. To investigate the relative impact of the ER and mitochondria in [Ca2+]i regulation together with the association on MN degeneration in G93A transfected SH-SY5Y cells, we used pharmacological agents as a specific activator of the ER-dependent Ca2+ release response [59], which not only selectively blocks Ca2+ uptake into ER/mitochondrial compartments but also triggers Ca2+ release from intracellular storage sites. Caffeine was purchased from Sigma-Aldrich chemie (Deisenhofen, Germany) and dissolved in water (25 mM). It was then diluted to the final concentration immediately before the experiment. We utilized KCl (K+) concentrations of 30 mM for depolarisation induced Ca2+ transients.

Results

Representative non-transfected SH-SY5Y neuroblastoma cells (parental cell line) stained with Fura-2 AM are shown in Additional file 2 [See Additional file 2]. Mitochondrial Ca2+ release responses were measured to verify the impact of mitochondrial uncoupler FCCP in normal SH-SY5Y cells. The application of FCCP for 2 min fairly inhibited mitochondrial Ca2+ and most of the cells had a proper distinguishable and characteristic fluorescence response (See Additional file 2]. In the cells matching set criteria, including a normal morphological appearance of a large soma and intense proper staining, we performed Ca2+ imaging 2–5 days after culture from WT-SOD1 and G93A transfected SH-SY5Y cells.

Calcium uptake by the mitochondria is accomplished by the mitochondrial calcium uniporter (MCU) located in the inner mitochondrial membrane [60]. The accumulation of mitochondrial Ca2+ may have a perceptible influence on the existence of a [Ca2+]i transient. This effect was studied by following the Ca2+ signal under healthy and deleterious mitochondrial conditions in the presence of FCPP. We found that, in G93A transfected SH-SY5Y cells, the Ca2+ influx (peak fluorescence) was diminished compared to WT transfected SH-SY5Y cells, which, peaked within 2–3 min and was followed by a baseline recovery in approximately 2 min. The G93A transfected cells took almost 5–6 min to achieve a baseline recovery. However, the cells transfected with G93A suffered from reduced [Ca2+]mito after depletion of mitochondrial Ca2+ stores by FCCP (Fig. 1).

Effect of high K+-evoked Ca2+ transient and its impact on FCCP-induced Ca2+ influx

To analyze the comparative efficiency of mitochondria as a Ca2+ sequestering organelle, FCCP was applied to evacuate mitochondrial Ca2+after the WT and G93A transfected cells had been exposed to an evoked Ca2+ load through a depolarizing stimulus of K+. First, the SH-SY5Y cells transfected with WT or G93A were stained with Fura-2 AM and exposed to 30 mM K+ for 30 sec, which was followed by a 3 min challenge with FCCP (Fig. 2). As shown in Figs. 2A and 2B, the peak amplitude of FCCP-evoked Ca2+ influx after depolarization was 0.2440 ± 0.0696 (N = 3, n = 20) and 0.1497 ± 0.0362 (N = 5, n = 20) for the WT and G93A transfected cells, respectively. Fig. 2C shows the comparative analysis of average calcium release.

Interaction between ER/mitochondria in differential Ca2+ store regulation by inhibition of the sarcoplasmic/endoplasmic reticulm Ca2+-dependent ATPase pump after pharamacological intervention

The relationships between mitochondrial calcium pools and those discharged by the Ca2+-ATPase inhibitor thapsi, an extremely tight-binding inhibitor of intracellular Ca2+ pumps which induces rapid Ca2+ release from intracellular stores by inhibition of the sarcoplasmic/endoplasmic reticulm Ca2+-dependent ATPase pump without inositol phosphate formation [57, 61] were studied in WT and G93A transfected SH-SY5Y cells to estimate reciprocal functional interplay between the ER and mitochondria. It was previously shown in SOD1G93A mice that release of ER based Ca2+ stores play minimal role and not an essential factors in the death mechanism of ALS vulnerbale MNs [62]. However, contrary to this study it was also shown that Ca2+ release from the ER contributes to neuronal cell death because the Ca2+release blocker, dantrolene, can protect neurons against bioenergetic failure and cellular damage [63]. While little is known about the exact ER/mitochondrial Ca2+ regulation mechanism in WT and G93A transfected cells, the restoration of ER function or attenuation of the secondary dysfunction induced by ER could present a new, highly promising mechansim for pharmacological intervention which could bring new ways to treat or minimize neuronal cell injury in the pathological states of ALS.

Analysis of the Ca2+ storing ability of ER and mitochondria in WT and G93A transfected cells was done using thapsi and FCCP. We show here that in fura-2-loaded cells, thapsi stimulated a slow plateau phase increase in cytoplasmic Ca2+ concentration in WT and G93A transfected cells. There was a significant quantitative difference between the ER and mitochondrial Ca2+ load in WT and G93A transfected cells; the Ca2+release response was high in the mitochondria of WT transfected cells. As shown in Fig. 3, the peak amplitude of mitochondrial Ca2+ release after application of FCCP plus thapsi was 0.2712 ± 0.0971 and 0.1276 ± 0.0287 where as peak amplitude of ER Ca2+ release after application of thapsi was 0.0412 ± 0.0152 and 0.02589 ± 0.0137 in WT and G93A transfected cells, respectively (Figs. 3A–C). The impact of thapsi on the peak amplitude of Ca2+ release was nominal compared with FCCP, indicating a lesser role for ER compared with mitochondria in Ca2+ regulation. Comparative details of the normalized fura-2 ratio in both WT and G93A transfected cells are summarized in Fig. 3C (N = 3, n = 15).

Interaction of ER/Mitochondria calcium stores regulation by activation of Ca2+ influx from the RyR-dependent Ca2+ stores

As shown in Fig. 4, caffeine led to a relatively slow and weak increase in the [Ca2+]i fluorescence signals, which was very similar for both WT and G93A transfected cells (N = 3, n = 14). We found that WT transfected cells (Figs. 4A and 4C) were affected by 5 mM caffeine with a small and slow increase in [Ca2+]i (F/F0 = 0.0471 ± 0.0190) and exhibited slightly higher kinetics than the G93A transfected cells ([Ca2+]i: F/F0 = 0.0353 ± 0.0120; Figs. 4B and 4C). This suggests that the caffeine-evoked ER-dependent Ca2+ release either makes only a minor contribution to mitochondrial mediated toxicity in G93A transfected cells, as reported in other cell types and animal models, or occurs upstream of the mitochondria.

The cells were treated with FCCP in the presence of caffeine, which resulted in the depolarization of the mitochondria (Fig. 4). We observed a rapid and high amplitude increase of [Ca2+]i, which exhibited a very distinct and differential response for WT and G93A transfected cells (N = 3, N = 14). WT transfected cells revealed that caffeine and FCCP together evoked a massive and comparatively fast increase in the Fura-2 fluorescence signals for WT (F/F0 = 0.1883 ± 0.0584, Figs. 4A and 4C) and G93A transfected cells (F/F0 = 0.1154 ± 0.0246, Figs. 4B and 4C).

As an uncoupler of mitochondria, FCCP collapses the mitochondrial membrane potential. In turn, this results in a rapid release of calcium from this store and a slower drop in ATP levels [64]. Impairment of the mitochondria with FCCP may block oxidative phosphorylation and has the potential to compromise oxidative phosphorylation; in this state, glycolysis provides the prime means of ATP synthesis. Previously, evidence had suggested that ATP depletion by FCCP in MNs leads to minimal effect on reverse cycle of uncoupling of oxidative phosphorylation ([62], S. Balakrishnan and B.U. Keller, unpublish data). We assume that the sole effect of FCCP on cells is the uncoupling of mitochondria and that this results might lead to a reduction of the proton gradient across the internal mitochondrial membrane. Oligo, an ATP synthase inhibitor, also depletes ATP but does not alter mitochondrial membrane potential and thus does not change calcium transport across the mitochondrial membrane. Oligo was added with FCCP to prevent any accelerated consumption of cellular ATP by the reverse mode of ATP synthase operation [65, 66].

To clarify this issue, we measure the impact of oligo (5 μg/ml) on FCCP-induced Ca2+ release in WT and G93A transfected SH-SY5Y cells loaded with fura-2. Very weak effects to those previously observed with the electron transport inhibitors (CN-) and azide were seen with oligomycin [19, 62]. As shown in Figs. 5A–C, impact of oligo's on the normalized peak fluorescence of fura-2 was 0.0454 ± 0.0175 and 0.0229 ± 0.0161 for WT and G93A transfected SH-SY5Y cells, respectively (Fig. 5C). The impact of oligomycin on the peak amplitude of fura-2 fluorescence was nominal and there was slight difference between WT and G93A transfected SH-SY5Y cells. However, the response to FCCP plus oligomycin in WT transfected cells (Fig. 5C) was almost 2.71-fold higher (F/F0 = 0.2245 ± 0.0727) compared with G93A transfected SH-SY5Y cells (F/F0 = 0.0827 ± 0.0304; Figs. 5A–C).

Discussion

Recent evidence suggests that abnormalities in cellular Ca2+ signaling are common features in the pathogenesis of a range of neurodegenerative disorders, including ALS [33]. It is well known that Ca2+ is one of the most relevant intracellular messengers essential in neuronal development, synaptic transmission and plasticity, and the regulation of various metabolic pathways in the brain. Further evidence for the involvement of a disruption in intracellular Ca2+ homeostasis was reported in cellular and experimental animal models; there was an absence of Ca2+ binding proteins, such as Calbindin-D28K and parvalbumin, in MN populations lost early in ALS [67]. Additionally, accumulation of Ca2+ into vacuoles in the mtSOD1 mice and low Ca2+ buffering in SMNs were also shown [68]. These findings agree with a quantitative comparison of Ca2+homeostasis where a low cytosolic Ca2+ buffering capacity acts as an important risk factor for degeneration. In contrast, an increase in the cytosolic Ca2+ buffering capacity could protect vulnerable MNs from degeneration [69, 35]. MNs store a larger amount of calcium in the mitochondria and disruption of mitochondrial Ca2+ uptake has a marked influence on both the peak amplitude of Ca2+ response as well as the clearance of [Ca2+]i.

Our interest in studying the role of Ca2+ regulation and the impact of mitochondrial inhibition in a cellular model of ALS is based on observations that mitochondria act as local calcium buffers, thus shaping the spatiotemporal aspects of [Ca2+]i. MN mitochondria have been shown to have a major percentage of Ca2+ sequestered intracellularly after influx through the plasma membrane [70, 71]. Our main objective was to characterize the contribution of the mitochondrial buffering of voltage-activated Ca2+ loads and how this Ca2+ regulatory mechanism is controlled. We observed that Ca2+ influx was diminished by approximately 1.86 fold in the presence of G93A compared to WT (Fig. 1). After exposure to FCCP, the capacity of G93A transfected cells to transport [Ca2+]i to the extracellular space or to intracellular storage sites was much lower than WT transfected cells and accounted for the increased vulnerability of cells possessing mutant G93A gene.

Interestingly, the depolarization-induced Ca2+ transient in the presence of intact mitochondria was smaller in amplitude than with G93A mitochondria; excess Ca2+ emerged in the second response and was prominent in WT cells (Figs. 2A–C). This reflects the variability in Ca2+ clearing mechanisms of ALS-vulnerable and non-vulnerable MNs and agrees with data obtained from motor nerve terminals [72], cultured MNs [25], and patch-clamped hypoglossal MNs [52] in which significant mitochondrial Ca2+ uptake following voltage-activated Ca2+ influx was demonstrated. We observed that the depolarization-induced stimulus not only interfered with the post-depolarization recovery of [Ca2+]i after FCCP challenge but also increased the peak amplitude of the FCCP-evoked Ca2+ influx by approximately 40 and 68% in WT and G93A cells, respectively (compare Figs. 1D and 2C). Therefore, it is evident that in the absence of mitochondrial Ca2+ uptake, the [Ca2+]i signal is stronger, and mitochondria can actively sequester Ca2+ during an on-going Ca2+ influx as a result of the opening of voltage-gated calcium channels. A significant difference in the Ca2+ accumulation by mitochondria in G93A and WT cells was observed and presumably due to G93A-mediated toxicity. The capacity of mitochondria to restore the Ca2+ buffering capacity of cell organelles is heavily compromised in G93A cells. The disturbance of Ca2+ trapping in intracellular storage sites and Ca2+ extrusion from the cells [41] may account for the reduced oscillations of [Ca2+]i we observed in G93A cells.

Molecular interaction underlying mitochondria-endoplasmic reticulum Ca2+ strores coupling was evaluated using thapsi and FCCP. We assume that both ER and mitochondrial intracellular pools participate in the generation of Ca2+ signals in SH-SY5Y cells shaping their spatiotemporal Ca2+ signals patterns. Thapsi- induced Ca2+release was significantly less than that evoked by FCCP in WT and G93A transfected cells, and its kinetics were more or less similar in both genotypes (Figs. 3A–C). The application of thapsi on SH-SY5Y cells causing separate Ca2+ release response was slightly more in WT transfected cells than in G93A cells, indicating that, in the context of the G93A transfection, the ER may slightly contribute to the motor dysfunction. Furthermore, the application of FCCP after emptying ER stores with thapsi resulted in a separate release event, evident from the bigger [Ca2+]i increase. This release was higher than the general Ca2+ release caused by FCCP without emptying ER in WT transfected cells but not in G93A altered cells, which suggests an uptake of the released Ca2+ from ER by mitochondria in WT transfected cells. This further indicates the explicit action of FCCP in our working model system and the existence of two separate intracellular Ca2+ stores, in which the ER seems to play a minimal role in buffering [Ca2+]i after Ca2+ loads are imposed in G93A transfected cells, and the ER is most likely not impaired during ALS-related motoneuron disease.

We found that ER of SH-SY5Y cells retained a very low amount of calcium (after Ca2+ release with thapsi for 6 min application) compared with mitochondria after Ca2+ elevation by FCCP (3 min) indicating its low efficiency to sequester Ca2+ in the WT and G93A transfected SH-SY5Y cells, which was slightly higher in WT (Figs. 3A–C). This indicates that the conventional mitochondrial Ca2+ storing function dominates ER Ca2+accumulation in these cells. It is noteworthy that thapsi has a relatively very weak effect on Ca2+ release in G93A transfected cells, indicating that G93A alteration might also result in defects in ER Ca2+ handling, which may perturb functional domain and contribute to neurodegeneration. These data suggest that the ER of SH-SY5Y cells does not play a significant role in regulating [Ca2+]i at the basal level or after imposed Ca2+loads in WT transfected cells; though minor ER contribution to the dysfunction of Ca2+ loads did not ruled out, suggesting that Ca2+dysregulation as a result of the G93A transfection anticipates mitochondrial impairment. Our hypothesis is strengthened by the fact that previously we have shown quite similar results in an animal model of SOD1G93A mice for fALS; where cyclopiazonic acid (a specific inhibitor of Ca2+-ATPase led to a relatively slow and weak increase of [Ca2+]i, which occurred with slightly higher kinetics in WT HMNs than the SOD1G93A mice HMNs [62]. This finding alongwith our results in WT and G93A transfected cells further explains that ER-dependent Ca2+release is a minor contributor to mitochondria-mediated toxicity in G93A transfected cells as previously reported in other cell types and animal models [73, 74]. Results indicate close coupling between ER and mitochondria in WT but not in G93A transfected cells which is probably impaired due to G93A transfection. The thapsi-releasable Ca2+ pool in WT and G93A transfected cells (Figs 3A–C) was quite similar in quantity and temporal quality release via RyR-sensitive Ca2+ pool by caffeine (see Figs 4A–C). This observations suggests that Ca2+ uptake by RyR-responsive pool is also sensitive to thapsi. These findings also indicate that thapsi increases [Ca2+]i by inhibiting Ca2+ uptake into multiple intracellular Ca2+ pools and by also promoting entry of extracellular Ca2+ dominantly in WT but not in G93A transfected cells. The exact molecular interactions defining the organization of mitochondria, ER and other Ca2+ sources varies among different cell types, are questions that remain unanswered but are interesting areas for future investigations.

We expect a decrease in cellular ATP levels during inhibition of mitochondrial electron transport by FCCP and this may affect release of [Ca2+]i. To test the impact of ATP depletion on regulation of [Ca2+]i, oligo (5 μg/ml) was added before FCCP application. Oligo does not influence [Ca2+]i heavily and left basal Ca2+ levels, as well as recovery times of Ca2+ transients, unaffected for upto several minutes (6 min application; Figs 5A–C) and therefore amplitude of evoke [Ca2+]i transients was clearly attributable to FCCP. Since application of oligo alone did not affect the [Ca2+]i transients therefore the effect of FCCP is clearly attributable to collapse of mitochondria, and not to inhibition of ATP-synthesis. We assume that the time course of ATP depletion by FCCP is slower but the increase of cytosolic calcium by FCCP is immediate and therefore there is no causal relationships have been demonstrated between fall in ATP level and increase of [Ca2+]i. These results are in agreement with those from studies of SOD1G93A mice in sensitivity of HMNs mitochondria to oligo [62].

Using the CCD camera imaging system, we clearly separated the dynamic profiles of [Ca2+]i and [Ca2+]mito. Using this method, we demonstrated that in WT cells, the FCCP-evoked increase in [Ca2+]i was mainly due to [Ca2+]i alone. However, in G93A cells, the FCCP-evoked [Ca2+]i was less, exhibited slower kinetics, and reduced [Ca2+]mito. This suggests that cells with G93A have a lower affinity for mitochondrial Ca2+ upload and likely represents severely disturbed and vulnerable mitochondria. In this study, for the first time, the simultaneous determination of [Ca2+]i and [Ca2+]mito in WT and G93A transfected SH-SY5Y cells, a culture model of motor neuron disease was achieved (Figs 6A–C). This data is in agreement with previous findings that G93A cells exhibit damaged and stressed mitochondria [6] and increased ROS [47].

Different intracellular pools participate in the generation of neuronal Ca2+ signals, shaping their spatio-temporal patterns, and the cell life-death cycle. Mitochondria, in the kinetic and hot spot hypothesis, and the ER, by different classes of channels with distinct properties and highly defined expression patterns, have been implicated in the regulation of [Ca2+]i in many systems [41, 42]. Caffeine was used to investigate ER-dependent Ca2+ release and we found that it evoked a small and slow increase in [Ca2+]i with slightly faster kinetics in WT cells than G93A cells. This further suggests that ER-dependent Ca2+ release has a minor contribution in the mitochondria-mediated toxicity of G93A. The rate of increase in Fura-2 fluorescence following caffeine application was slower and did not resume the baseline, which is likely due to the slow activity of the mitochondrial Na+/Ca2+ exchanger, the major pathway for mitochondrial Ca2+ efflux [75]. Observations of different pharmacological conditions support the concept that the presence of G93A severely disrupts mitochondrial Ca2+ regulation. It is also interesting to note that the rate of increase in Fura-2 fluorescence signals following caffeine application was slightly slower compared to FCCP-evoked signals and did not achieve the baseline within a few minutes. This suggests that the dye accumulates in the mitochondria and the mitochondrial uptake and release event is prominently operated by Ca2+ efflux rather than by the store-operated Ca2+ uptake and release phenomenon. Our fluorescence system may be a valuable tool to determine mitochondrial and Ca2+-related defects during G93A-mediated MN degeneration, which closely parallels the incidence of neuronal death in G93A transfected SH-SY5Y cells. Quantitative values of normalized Fura-2 fluorescence signals after different drugs interventions are compared in Table 1.

Table 1

Differential peak amplitudes of [Ca2+]i release from different compartment of WT and SOD1 transfected SH-SY5Y cells after multidrugs intervention.

Despite rigorous research since Charcot's description more than 130 years ago, the molecular abnormalities leading to the damage of specific MNs in ALS are still unknown. The selective vulnerability of MNs in ALS-related disease and associated cell culture models is closely linked to exceptional Ca2+ signaling mechanisms that are part of the physiological cell function, but seemingly also enhances the risk of Ca2+ homeostasis disruption and mitochondrial dysfunction in vulnerable cells. Earlier studies suggested that uncontrolled Ca2+ entry and inefficient calcium sequestering cause selective damage leading to the formation of vacuoles derived from the degenerating mitochondria in the MNs of the mouse model of ALS [12, 76, 77]. In contrast to most other neurons, MNs have a low Ca2+-buffering capacity due to the low expression of Ca2+-buffering proteins and a high number of Ca2+-permeable AMPA receptors resulting from low expression of the GluR2 subunit. The combination of these two properties seems to be intrinsic to MNs and is most likely essential for their normal function. However, under pathological conditions, MNs may become over-stimulated by glutamate and overwhelmed by Ca2+; though, whether downstream pathways activated by the intracellular Ca2+ increase are different in MNs compared to other neurons is not yet known.

Conclusion

Our experiments identified specialized Ca2+ homeostasis characterized by low cytosolic Ca2+ buffering in which mitochondria play a major role in the regulation of [Ca2+]i transients in vulnerable MNs. Low cytosolic Ca2+ buffering enhances the role of low affinity organelle buffers, such as mitochondria, in the cell. For example, large and long-lasting Ca2+ domains around influx sites enhance the risk of toxic Ca2+ accumulations and subsequent activation of Ca2+-dependent neurodegenerative pathways under excitotoxic conditions. Indeed, a strong contribution of mitochondria, as opposed to ER, Ca2+ uptake to the buffering of Ca2+ profiles was recently demonstrated [19, 52, 62]. There are two reasons that the prominent role of mitochondria in the regulation of moderate Ca2+ loads in MNs has important implications for pathological conditions such as ALS. First, the amount of Ca2+ taken up by the mitochondria is probably higher in MNs than other cell types [25, 78]. These high Ca2+ loads enhance the risk for ROS generation, which may play a major role in initiating the death cycle resulting in MN degeneration [29]. Second, our experiments provide evidence that cytosolic Ca2+ depends on intact mitochondrial Ca2+ uptake. Thus, when mitochondrial Ca2+ uptake is disturbed, as was seen in G93A cells, MNs are directly put at risk of accumulating basal Ca2+ levels during repetitive oscillations. Additionally, the decreased ability to limit Ca2+ transient amplitudes in the cytosol and, in particular, local domains when mitochondria are depolarized enhances the risk of initiating Ca2+ dependent neurodegenerative pathways leading to cell death. A summary of the identified mechanisms is given in Fig. 7; in vulnerable MNs, the calcium buffering machinery is represented by the predominance of mitochondria and calcium binding proteins.

Figure 7

Low Ca2+buffering and excitotoxicity under physiological stress and pathophysiological conditions in motor neuron (MNs). Low Ca2+ buffering in amyotrophic lateral sclerosis (ALS) vulnerable hypoglossal MNs exposes mitochondria to higher Ca2+ loads compared to highly buffered cells. Under normal physiological conditions, the neurotransmitter opens glutamate, NMDA and AMPA receptor channels, and voltage dependent Ca2+ channels (VDCC) with high glutamate release, which is taken up again by EAAT1 and EAAT2. This results in a small rise in intracellular calcium that can be buffered in the cell. In ALS, a disorder in the glutamate receptor channels leads to high calcium conductivity, resulting in high Ca2+ loads and increased risk for mitochondrial damage. This triggers the mitochondrial production of reactive oxygen species (ROS), which then inhibit glial EAAT2 function. This leads to further increases in the glutamate concentration at the synapse and further rises in postsynaptic calcium levels, contributing to the selective vulnerability of MNs in ALS.

Abbreviations

ALS:

amyotrophic lateral sclerosis

AM:

acetoxy methyl ester

CCD:

charge cooled device

DMEM:

Dulbecco's modified Eagle medium

DMSO:

dimethyl sulfoxide

ER:

endoplasmic reticulum

F:

fluorescence

fALS:

familial amyotrophic lateral sclerosis

FCCP:

Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone

FCS:

fetal calf serum

FMNs:

facial motoneurons

HMNs:

hypoglossal motoneurons

hALS:

human amyotrophic lateral sclerosis

MCU:

mitochondrial calcium uniporter

MN:

motoneuron

mtSOD1:

mutant Cu/Zn superoxide dismutase1

oligo:

oligomycin

ROI:

regions of interest

ROS:

reactive oxygen species

RPMI 1640:

Roswell Park Memorial Institute medium

RyR:

ryanodine receptor

SD:

standard deviation

SOD1:

Cu/Zn superoxide dismutase1

TPP+:

Tetraphenylphosphonium salt

thapsi:

thapsigargin

[Ca2+]i:

cytosolic calcium

[Ca2+]mito:

mitochondrial calcium

WT:

wild type

Δψm:

mitochondrial membrane potential.

Declarations

Acknowledgements

We would like to thank Drs Saju Balakrishnan, Friederike von Lewinski, Michael Müller, Eike Schomburg and Diethelm Richter for their valuable discussions. This research was supported by grants from the Bernstein Center for Computational Neuroscience (BCCN), Göttingen, BMBF project Bioprofile 0313610 A, the consortium EU – Eranet ERMCC and Else Kröner-Fresenius-Stiftung.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

MKJ performed the experiments, analyzed the data, and wrote the initial version of the manuscript. WDZ, MG and RN established the primary cultured human SH-SH5Y cell line and maintained them during the course of the project. Cell lines expressing either wild-type (Wt) human SOD1 or the G93A mutants of this enzyme were produced by AF and MTC. CL, AZ, MTC and RN participated in the design of the study and participated in the organization of financial support. BUK is the principal investigator of the project. Besides providing laboratory support and supervision, he is primarily responsible for financial support and project management. All authors read and approved the final manuscript.

Copyright

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.